PROJECT SUMMARY Despite major advances in targeted and immune therapies for melanoma, most patients experience relapses or do not respond adequately. Acquired and intrinsic resistance to these therapies in both patient biopsies and cell lines have been associated with a unique undifferentiated cell state characterized by low levels of MITF, the master regulator of melanocyte development, and high levels of the AXL tyrosine kinase. This cell state is reported in a high fraction of melanomas exhibiting de novo resistance to BRAF inhibition and in 50-100% with acquired resistance. It is also a prominent feature of anti-PD1 resistant melanomas. Single-cell RNA-seq demonstrated that all melanomas, even high-MITF ones, contain low-MITF subpopulations that are positively selected by targeted therapy. To eradicate this population, AXL itself is not a therapeutic target, based on published tests of multiple inhibitors and shRNAs. We therefore interrogated genetic dependencies using Project Achilles?an unbiased genome-scale loss-of-function screen?and found that low-MITF/high-AXL melanomas exhibit a striking dependency, not on AXL, but on LSD1, a lysine-specific histone demethylase that has been implicated in oncogenic processes. We validated LSD1 dependence in multiple melanoma lines using both genetic and pharmacologic inhibition. Analysis of melanomas in the TCGA and CCLE databases for candidate LSD1 target genes mediating its dependency revealed three candidates that LSD1 selectively modulates in low-MITF melanomas. The first of these, NDRG1, was shown to be required for LSD1-targeted lethality. Concordance of these LSD1-target expression patterns is seen across both cell lines and tumors (TCGA). We also show that SP2509, an LSD1 inhibitor related to a compound in clinical development, is highly lethal to multiple low-MITF melanomas, an effect that is dependent upon (and modulated by) the low-MITF state. Deeper analysis of the Achilles database identified multiple subunits of CoREST (known LSD1 co- repressive complex) as strong dependency factors, phenocopying selective lethality of low-MITF/high-AXL melanomas and suggesting a mechanistic connection to LSD1-dependency. In Aim 1, we will test the hypothesis that therapeutic resistance of low-MITF melanomas can be mitigated by combining targeted or immune therapies with LSD inhibition in early passage melanoma cell lines and PDX. Our preliminary results demonstrate profound cooperative lethality by combining genetic or pharmacologic LSD1 inhibition with BRAF inhibitor in vitro and in xenografts. We will test the ability of LSD1 inhibition to antagonize the emergence of resistance in high-MITF melanoma, as well as to cooperate with agents that actively suppress MITF expression. In Aim 2, we will mechanistically examine functional roles of four candidate mediators of LSD1 dependency and use genomic approaches to systematically scrutinize the low-MITF state, identifying potential pharmacodynamic markers and mediators of LSD1 dependency. These findings provide a unique opportunity to reveal mechanistic insights and therapeutic opportunities for important treatment-resistant patient subsets.